To analyze and determine the quality of functioning in different components of GHRH-GH-IGF1 axis in children with Down syndrome (DS).Systematic review and mini meta-analysis of the literature.A search was performed in PubMed, Embase, Scopus, and PsycINFO through August 2022. Eligible studies included pediatric patients with DS who had undergone any laboratory evaluation of the GHRH-GH-IGF1 axis. Two reviewers independently screened articles for eligibility. Results of each type of test were weighed together in patients both with and without DS and were pooled using a random effects meta-analysis.In total, 20 studies assessed the GHRH-GH-IGF1 axis function. A defect in three major components of GHRH-GH-IGF1 axis was found in a significant proportion of pediatric DS patients.A significant portion of short-stature pathogenesis in children with DS is associated with impaired GHRH-GH-IGF1 axis function. The main pathogenic route examined is the existence of interference in one of the “stations” along the GHRH-GH-IGF1 axis. The GHRH-GH-IGF1 axis consists of a chain of events that start with the release of growth hormone-releasing hormone (GHRH) from the hypothalamus. GHRH then travels to the pituitary gland, where it stimulates the release of growth hormone (GH). GH then travels to the liver, where it stimulates the production of insulin-like growth factor 1 (IGF-1). IGF-1 is a hormone that plays a critical role in growth and development.The involvement of GHRH-GH-IGF1 axis in the pathogenesis of short stature in DS children is strongly suggested by growth retardation having been most marked between the ages of 6 and 24 months, the same period that growth hormone normally becomes the main regulator of growth  guidelines . No form2.2.The scope of data reporting in a large part of the original works did not allow for a full quality and risk of bias assessment to be carried out on the individual original studies, therefore no individual quality assessment was carried out.We used ROBIS, a tool for assessing quality and risk of bias in systematic reviews. The tool is completed in three phases: assess relevance , identify concerns with the review process by 21 questions divided to 4 domains, and judge risk of bias in the review. It is the first rigorously developed tool designed specifically to assess the risk of bias in systematic reviews .2.3.Eligible studies included focused on the assessment of the GHRH-GH-IGF1 axis function in pediatric DS patients and reported results of at least one of the following tests: growth hormone stimulation test, 12- or 24-hour integrated GH concentration test, IGF-1 level assay, IGF-1 generation test, calculated bind GHBP (growth hormone binding protein)/ total GHBP ratio, or calculated GH Radioreceptor assay (RRA) / immunoradiometric assay (IRMA) ratio. Both comparative as well as single-arm studies were considered, as were single case reports. 2.4.Data extraction included a full description of participants enrolled including their gender and age. Foreign language articles were translated by multilingual reviewers. The main outcome extracted from studies for mini meta-analysis was the percentage of patients with pathologic test results.2.5.In order to assess heterogenicity, we used a random effects meta-analysis. We assessed the degree of inconsistency in the results between studies using the I² statistic. This statistic explains the proportion of inconsistency between studies that cannot be explained by chance alone and is likely due to real differences in the population or the conduct of the studies .Results of each type of test were weighed together in patients both with and without DS and were pooled using a random effects meta-analysis. We used a Chi-square test to evaluate the differences between test results of DS patients with those of control groups. Publication selection bias could not be calculated due to missed relevant data.3.3.1.An initial search of the literature yielded 281 publications and 20 eligible studies . Twenty The percentage of pathological results among DS patients ranged between 0% and 25% in studies that examined the arginine stimulation test , 10, 17,3.2.Risk of Bias of systematic review was evaluated according to ROBIS. Phase 2 include 3 essential domains: identification and selection of studies, data collection and study appraisal, synthesis, and findings. Thirteen signaling questions in three domains were corresponded to “low risk of bias” while 3 signaling questions, 2 in domain 3 and one in domain 4, were classified as “no information” and hence corresponded to “unclear risk of bias”.Comparisons of pathological results in percentage terms between DS and non-DS children for each test as presented in p value <0.001.Weighing the total results for all tests on DS children cumulatively yielded 40% pathological results compared to 0% in the non-DS control groups . The staA forest plot of standardized mean difference (SMD), and their 95% CI and weights for the different combined test results is presented in p-value <0.001. I-squared was 98.6%. The estimate of between-study tau-squared variance was 4.862 with a p-value <0.001.Using random effect analysis, chi-squared heterogeneity was 567.47 with a 3.2.1.Hypothalamic function was assessed by 12-h  or 24-h integrated GH concentration and by GH stimulation tests that can be classified to “hypothalamus-mediated” and “pituitary-mediated”.3.2.2.p-value of <0.02. Four such tests were conducted in DS patients. The mini meta-analysis of all the detailed and reported hypothalamus-mediated GH stimulation tests –20, 22, 3.2.3.The mini meta-analysis of all detailed reported 12-h  or 24-h integrated GH concentration reveals a very high (93%) pathological fraction , 4, indi3.2.4.p-value of <0.0001, 0.000192, and 0.077, respectively.GH releasing effect was studied in 12 DS patients showing that the response to hexarelin is normal and is similar to GHRH plus pyridostigmine, slightly higher than GHRH alone, and much higher than clonidine, levodopa, and insulin stimulation tests , with p-3.3.There was a difference between “endogenous” and “exogenous” IGF-1 generation tests. A normal reaction to the “exogenous” GH-induced IGF-1 generation test , 17, in 3.3.1.The mini meta-analysis of the detailed and reported IGF-1 level in DS pediatric patients, according to an acceptable age-dependent normal range , 24–27, 4.The picture that emerges from this systematic review and mini meta-analysis is that the GHRH-GH-IGF1 axis is impaired in children with DS. There is a well-established impression of a hypothalamic impairment expressed in a combination of quantitative production and functional neurosecretion disorders. Apparently, a significant portion of the pathogenesis of short stature in children with DS is associated with impaired GHRH-GH-IGF1 axis function, as evident from the results of the various tests.4.1.A variety of findings supports the hypothesis that the hypothalamus plays a significant part in the short stature among children with DS , specifi4.1.1.Insulin, levodopa, and clonidine tests revealed much more pathological results than the arginine test. This finding suggests involvement of the alpha-adrenergic neurotransmitter GHRH-mediating pathway and may point towards the original disturbance location.4.1.2.The significant difference between GHRH stimulating test results and ITT, Levodopa, Clonidine, and Arginine stimulation tests' results indicates normal or close to normal pituitary global function and supports hypothalamic dysfunction.4.1.3.The basis for understanding clinical disorders in the neuro-regulation of GH secretion is derived from the complexity of the CNS hypothalamic–pituitary axis. Studies in animals and humans demonstrate anatomic, physiological, and pharmacological evidence for neurosecretory control of GH secretion. The observation of a defect in the neuroregulatory control of GH secretion in CNS-irradiated humans and animals led to the hypothesis of a disorder in neurosecretion, GHND (growth hormone neurosecretion dysfunction), as a cause for short stature . The ver4.2.DS subjects seem to lack the physiological switch from the production of fetal somatomedins and IGF-2 which are growth hormone independent, to IGF-1, which is growth hormone dependent . In ligh4.2.1.Liver and brain IGF-1 receptors are normal in DS fetuses . The res4.3.A high heterogenicity index may be explained by the fact that, although the tested outcome- the rate of pathological tests- was uniform, this systematic review compares different tests that evaluate different “stations” along the hormonal axis. Nevertheless, it is possible to clearly identify the absolute majority of the results relatively close to the 0 line with the exception of 2 tests: GHRH and ICGH. In both cases, the total number of participants is relatively small compared to the other tests- a fact that increases the chance of sampling error. Although the total number of participants in those two tests is relatively small, this is the maximum number of participants reported in the literature to the best of our knowledge.4.4.The findings of this review indicate a defect in three major components of the growth hormone axis in a significant proportion of pediatric DS patients. The first one is the quantitative capacity of the hypothalamus–pituitary axis, the second is the qualitative capacity of this axis, and the third is the reduced bioactivity of endogenous GH in some DS patients.4.4.1.The results of this review quite compellingly indicate that the GHRH-GH-IGF1 axis is impaired in children with DS and significantly strengthen the view that GHRH-GH-IGF1 axis assessment should be considered in short DS children.According to our findings, assessing GHRH-GH-IGF1 axis function in DS children with the help of only classic stimulation tests may mislead the clinician. These tests mainly examine the quantitative capacity of growth hormone secretion. Although we have shown that about a third of children with DS suffer from an abnormal quantitative capacity and are formally diagnosed as having growth hormone deficiency based on two pathological stimulation tests, other disorders in the GHRH-GH-IGF1 axis such as GH neurosecretory dysfunction or bio-inactive GH are likely to be missed if we rely solely on growth hormone stimulation tests.It is possible to consider performing a nocturnal spontaneous GH secretion test for evaluating the growth hormone axis in children with DS. This is a test that may sometimes be incorporated into clinical practice .Exogenous growth hormone treatment is expected to provide a response to all three main issues identified in this study. In contrast to growth hormone receptor resistance and more distal disturbances such as IGF-1 receptor deficiency, the administration of proper recombinant growth hormone circumvents both the quantitative and qualitative disorders in the production and secretion of growth hormone, as well as defects in the endogenous protein structure. However, such therapy should undergo formal testing in prospective long term clinical studies.


The application of many currently evaluated macromolecular contrast agents for magnetic resonance imaging (MRI) has been limited because of their bio-incompatibility and toxicity. The aim of this study is to synthesize and characterize a new micelle-based TPGS gadolinium chelate as a biocompatible MRI contrast agent for prolonged blood circulation time and good tumor imaging contrast. The TPGS-gadolinium conjugate was prepared through the conjugation between TPGS-SA and bifunctional L-NETA-Gd chelate. The conjugate was characterized with regard to molecular weight, critical micellar concentration and particle sizes, cellular uptake, and in vitro cell MRI. Distributions of the MRI contrast agent in various organs were determined via intravenous injection of the agent into mice bearing xenograft tumors. The successfully prepared TPGS-L-NETA-Gd micelle exhibited improved cellular uptake in HepG2 cells and xenografts and high in vivo safety. Distributions of TPGS-L-NETA-Gd in mice showed enhanced cellular uptake up to 2 h after the contrast agent injection. Its in vitro and in vivo properties make it a favorable macromolecular MRI contrast agent for future in vivo imaging. Nowadays, various imaging technologies, such as ultrasound, computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT), are used for the evaluations of diagnosis and therapy ,2. AmongTM), have been utilized preclinically for cell labeling in order to enable tracking and monitoring of stroke lesions or tumor growth in vivo +: 630.1648; Found 631.1720.Gadolinium oxide  was added to a solution of 1 g of bifunctional chelator L-NETA (2 mmol) in 5 mL of DI water. The reaction mixture was heated to 100 °C and reacted for 6 h. Over that time period, the solid oxide dissolved and the pH increased from 2 to around 7. The xylenol orange test was applied to determine the presence of free GdN-dicyclohexylurea (DCU) and then dialyzed using MWCO 500 membrane in water for 24 h to remove DMSO and unconjugated L-NETA-Gd. The resulting solution was lyophilized to obtain TPGS-L-NETA-Gd as a waxy solid. Estimated mass: 2300 ± 500; Found: 2300.1999 ± 200 .TPGS-SA , dicyclohexylcarbodiimide , and DMAP  were dissolved in 10 mL of dimethyl sulfoxide (DMSO), and the resulting mixture was incubated at room temperature for 1 h. L-NETA-Gd  and triethylamine (TEA)  were added to the reaction mixture and further incubated for 20 h at room temperature. The reaction mixture was filtered to remove N,3 and dried to a thin film in a round-bottomed flask on a rotary evaporator under reduced pressure for 30 min at 45 °C. After it was evaporated by rotary evaporation, the dried lipid mixture was then rehydrated in 2 mL of phosphate buffer (pH 7.4) at 60 °C for 1 h under flowing nitrogen. The resulting suspension of vesicles was extruded through the 200 nm and 100 nm pore size polycarbonate membranes at least five times using a Lipex extruder .To prepare TLNm, TPGS-L-NETA-Gd was dissolved in CHCl2 incubator.HepG2 and LO2 cells were cultured with high-glucose DMEM supplemented with penicillin, streptomycin, and 10% FBS in a 37 °C and 5% COBALB/c nude, female  mice and Kunming mice were purchased from Beijing Huafukang Bioscience Technology Co., Ltd. . The animals were cared for under an animal use protocol approved by Huazhong University of Science and Technology. The study was approved and conducted according to the rules set forth by the Institutional Animal Care and Use Committee (IACUC) of Tongji Medical College, Huazhong University of Science and Technology .6 cells per mouse) were suspended in 100 µL of PBS and injected subcutaneously into BALB/c nude mice. When the tumor size reached about 5 mm in diameter after about 10 days, the nude mice were used for the in vivo study.To induce a tumor, HepG2 cells  at an accelerating voltage of 100 kV.−4 M solution of pyrene in DCM was added in every vial and mixed well. The DCM was left to evaporate for 24 h in order to form a pyrene film in the vial. Finally, 15 mL of Milli-Q water was added to the vials to obtain a final pyrene concentration of 6.0 × 10−7 M for each vial and T-L-Gd solutions with concentrations ranging from 0.0002 mg/mL to 0.2 mg/mL. The solutions were kept on a shaker at 37 °C for 24 h to reach equilibrium before the fluorescence measurement. Fluorescence spectra were recorded on a luminescence spectrometer at room temperature. The excitation spectra were scanned from 300 to 350 nm at the emission wavelength of 373 nm. Excitation and emission bandwidths were 5 nm and 10 nm, respectively. The fluorescence intensity ratio of I342/I338 was analyzed as a function of micelle concentration.The CMC of micelles in water was determined with a fluorospectrometer using pyrene as a hydrophobic fluorescence probe . Briefly5 cells/well and incubated for 24 h with high-glucose DMEM supplemented with penicillin, streptomycin, and 10% FBS in a 37 °C and 5% CO2 incubator. The concentrations of TLNm and Gd-DTPA were diluted into 20 μg/mL (with Gd content) and were added to the medium for 8 h, 4 h, 2 h, 1 h, or 30 min. In order to measure the uptake of TLNm and Gd-DTPA, cells were washed with PBS 3 times and then trypsinized, collected, and digested in aqua regia. The concentration of the Gd (III) was determined via ICP-AES .To analyze the uptake of TLNm and Gd-DTPA in vitro, HepG2 cells were plated on 24-well plates at a density of 1 × 106 cells /tube). The cells were subsequently incubated for 2 h at 37 °C with 5% CO2. Cells in each EP tube were transferred at 1500 rpm and washed with PBS (3 × 1 mL). Cells were resuspended with 500 μL of 1 × PBS (PH = 7.4). A multi-slice SE sequence was used.For the cellular MRI, HepG2 cells were seeded into culture dishes with 10 mL of culture medium. When the cells reached 80% confluence, the medium was replaced with fresh medium containing different samples (TPGS-NETA-Gd micelles and Gd-DTPA at the same gadolinium concentration of 20 μg/mL). Meanwhile, another dish replaced with only fresh medium was selected as a control. After 2 h of incubation, the medium was removed and washed with PBS three times. Then, cells were harvested after the treatment with trypsin and centrifugation. The resulting cells were transferred to 200 μL Eppendorf (EP) tubes and then centrifuged at 1000 rpm to obtain a compact pellet at the bottom of the tube and directly measured for MRI. In the in vitro experiments, HepG2 cells or LO2 cells were grown in different concentrations of TLNm and Gd-DTPA complex  supplemented with 10% fetal bovine serum FBS) or PBS, in 1.5 mL EP tubes . Livers and kidneys were dissected, fixed in 4% neutral paraformaldehyde solution, embedded in paraffin wax, and further sliced and stained with hematoxylin and eosin (H&E) for microscopic observation.g for 10 min. Microplate assay was used for the determination of aspartate aminotransferase (AST) and alanine aminotransferase (ALT).TLNm was injected into Kunming mice at 0.1 mmol Gd/kg and, 5 days later, blood was sampled by cardiac puncture and collected in EDTA tubes. Plasma samples were separated by centrifugation at 2000× 6 cells per mouse) into the right hind flank of BALB/c female mice. The biodistribution of TLNm was determined at 0.5 h, 1 h, 2 h, and 4 h post-injection in comparison with Gd-DTPA. The contrast agents were administered at a dose of 0.1 mmol Gd /kg (TLNm concentration of 290 μg/mL) via tail vein injection. ICP-AES was used to measure the content of gadolinium in organs. Before the analysis, organs were dehydrated via freeze-drying. Tissue samples were crushed, then digested and mineralized with aqua regia (1 mL for each organ) at room temperature for 48 h. Finally, the solution was fixed in a 2 mL capacity bottle.A xenograft mouse model was generated by subcutaneous injection of HepG2 cells . Multiple groups were compared using a one-way ANOVA with Dunnett’s post-test. A value of p < 0.05 was considered significant and p < 0.01 was considered highly significant.The comparison of two groups was performed using Student’s 3. The absence of free Gd3+ was confirmed by the xylenol orange test. A solid white powder was formed after the lyophilization. The MALDI-TOF mass results with multiple peaks of the isotopes of gadolinium confirmed that L-NETA-Gd was formed. In order to conjugate TPGS with L-NETA-Gd, a succinic acid linker was introduced to TPGS. With the amide bond formation between TPGS-SA and L-NETA-Gd, TPGS-L-NETA-Gd was successfully prepared and purified using dialysis to remove the salt and unreacted L-NETA-Gd. The masses of TPGS-SA and TPGS-L-NETA-Gd were tested using MALDI-TOF, and the spectra shown in The synthesis of TPGS-L-NETA-Gd was achieved smoothly via a multistep synthesis A. To ancCMC is a vital property of micelles, affecting their stability and drug-loading efficiency for drug delivery. Thus, before further evaluation, the CMC of TLNm was determined using a traditional method based on a hydrophobic fluorescence probe pyrene . As showAfter determining the CMC, the TPGS-L-NETA-Gd micelle (denoted as TLNm) was subsequently diluted in PBS and passed through a 0.22 filter. The particle size distribution of TLNm was characterized by a dynamic light-scattering (DLS) technique. As shown in To determine the cell uptake property of TLNm, HepG2 cells were used, and the results were compared with those of Gd-DTPA A. The upTo investigate the ability of micelles to enhance T1-weighted contrast of cell populations, MR images shown in B and theTo determine whether TLNm can be delivered into a tumor in vivo, we examined the distribution of TLNm in HepG2 tumor-bearing nude mice. The animals were sacrificed at different time points after the injection of TLNm and Gd-DTPA, respectively, and the amounts of gadolinium in tissues were analyzed using ICP-AES. As shown in To further investigate the in vivo safety of TLNm, Kunming mice were executed 5 days after the injection of TLNm, Gd-DTPA, and PBS (control group), respectively. There were no significant inflammatory lesions or tissue damages in major organs (liver and kidney) compared to the control group. There was also no significant difference in weight changes among the three groups A, and thIn the current study, a gadolinium-containing micelle based on an FDA-approved material, vitamin E TPGS, was prepared using L-NETA as a chelating agent. The preparation and characteristics of the TPGS-L-NETA-Gd micelles were studied successfully. The CMC value of TLNm was determined to be 4.3 µg/mL, which is comparable to that of TPGS. The spherical morphology and monodispersity of TLNm were also confirmed via TEM, and the size was measured to be nearly 25 nm. In vitro cell-based assays confirmed the introduction of TPGS significantly increased the cellular uptake of gadolinium and showed obviously enhanced contrast compared to Gd-DTPA. The prepared TPGS-L-NETA-Gd micelles also exhibited high in vivo safety, and improved uptake in HepG2 cells and xenografts, rendering them potential MR contrasting agents for enhanced tumor imaging.


To investigate the clinical and radiographic outcomes of EXP-IFC in single-level MIS-TLIF.p < 0.05 in univariate analysis.This study included patients aged ≥18 years who received a single-level MIS-TLIF procedure with at least 1 year of follow-up. Outcome measures: clinical features, preoperative and neurological complications. Imaging analysis included disc height (DH) restoration, surgical and contralateral side foraminal height (FH), lumbar lordosis angle (LL), segmental lordosis (SL). Visual analog scale (VAS) score for low back pain (VAS-LBP) and leg pain (VAS-LP), Oswestry Disability Index (ODI) and Japanese Orthopaedic Association (JOA) score were used to evaluate clinical outcomes. Statistical analysis was performed using independent sample t-test and sample t-test. The significance was set to p < 0.001). The mean change in FH of operative and contralateral sides were observed to be significantly higher for the patients with EXP-IFC at 1 year follow-up , but changes in LL and SL were not significantly different. At the last follow-up, we did not find any significant difference in the fusion rate between the two groups.A total of 62 patients undergoing single level MIS-TLIFs between January 2017 and January 2019 were included, with 32 NE-IFC 46.9% female, mean age 54.86 ± 11.65, mean body mass index (BMI) 24.59 ± 3.63) and 30 EXP  with no significant differences in demographics. There were no significant differences between two groups in Operative time (OT), Estimated blood loss (EBL) and Length of stay (LOS). No significant differences were found in VAS-LBP, VAS-LP, JOA and ODI in post-operation and the last follow-up between the two groups. The imaging outcome demonstrated that the mean increase in DH was significantly greater for the patients with EXP-IFC than those with NE-IFC group at 1 year follow-up (8.92 ± 0.51 mm EXP-IFC vs. 7.96 ± 0.96 mm NE-IFC, Our results indicated that there may be no significant difference in short-term clinical outcomes between EXP-IFC and NE-IFC, but the use of EXP-IFC in MIS-TLIF can provide a significant restoration of disc height, and neural foraminal height compared to NE-IFC. Lumbar degenerative diseases are common in the elderly and often cause pain, disability, and poor quality of life . With thIn recent years, it is suggested that lumbar spine surgery should be performed on the basis of complete decompression to minimize the damage to the stable structure of the spine . With thHowever, there is still controversy concerning the ability of EXP-IFC to restore sagittal alignment and correction of radiographic parameters , 12. WitThe purpose of this study was to compare the clinical outcomes and restoration of LL, SL, DH and FH in patients who had underwent MIS-TLIF with either NE-IFC or EXP-IFC one year after surgery.A single-center retrospective analysis of adult patients (≥ 18 years old) undergoing selective single-level MIS-TLIF from January 2017 to January 2019 was conducted. Patients met the following inclusion criteria: (1) a minimum 1 year follow-up (2) diagnosis of single-level lumbar degenerative disease diagnosed by MRI; (3) no previous fusion history; (4) surgery performed by the same group of doctors; (5) the use of expandable and non-expandable fusion cages. Exclusion criteria were as follws: (1) severe degeneration in adjacent segments; (2) lumbar spondylolisthesis and severe bony spinal canal stenosis; (3) multilevel disease; (4) history of lumbar fracture, infection, deformity, tumor and severe osteoporosis; (5) incomplete clinical and imaging data.This study was approved by Medical Research Ethics Committee of Shanxi Bethune Hospital and informed consent was waived due to the retrospective nature of the study.In this study, the choice of fusion cage type was based on the optimal height according to preoperative imaging data. The height of fusion cage should be slightly larger than the preoperative intervertebral height by 20%, the length of the cage should be approximately 4 mm shorter than the anteroposterior diameter of the vertebral body. The cage is made of titanium alloy. Its height is between 8 cm and 13 cm and can be adjusted to the right position according to the surgical needs . All opeData on demographic, clinical characteristics and outcomes were obtained from the patients' electronic medical records. Demographic data included age, body mass index (BMI) and gender. Surgical parameters included operation time, estimated blood loss (EBL) and length of stay (LOS).The visual analogue scale (VAS) for low back pain (LBP) and leg pain (LP), the Oswestry disability index (ODI) and the modified Mac Nab standard were used to evaluate the clinical effect. Japanese Orthopaedic Association (JOA) score was used to evaluate the improvement of neurological function . An imprThe radiographic parameters included disc height (DH), foraminal height (FH), segmental lordosis (SL) and lumbar lordosis angle (LL) for each surgically treated level. DH was defined as the average of height of anterior edge of upper and lower vertebral body and height of posterior edge of vertebral body in responsible space; FH was defined as the vertical distance between the lowest edge of pedicle of upper vertebral arch and the highest edge of lower vertebral arch of responsible level; SL was defined as the angle between the superior and inferior endplates composing the disk space; LL was defined as the angle between the lower endplate of the L1 body and the sacral plate. Standard lateral lumbar x-ray examinations were performed pre-operatively, post-operatively and at 1 year follow-up. All these parameters were measured by Surgimap Spine Nemaris Inc (Version: 2.2.13.1). Two experienced orthopaedic surgeons who were not involved in the study measured the data on lumbar x-ray images and recorded the average of the measurement results.The fusion was determined by lumbar imaging  .p < 0.05.IBM SPSS Statistics Grad Pack 27.0  was used for analysis. Measurements following a normal distribution are expressed as the mean ± standard value p = 0.230). For the patients with NE-IFC, 54.6% were female, the mean age was 54.86 ± 11.65 years old, the average BMI was 24.59 ± 3.63. In the EXP-IFC group, 48% were female, the mean age was 58.32 ± 12.99 years old, with an average BMI of 24.45 ± 2.67. All cases were followed up for 1 year after operation to meet the standard of osseous fusion. At the 1 year follow-up, the bone fusion rate was 93.8% in NE-IFC group and 96.7% in EXP-IFC used group with 1 case not achieving osseous fusion, according to x-ray.This study included 62 patients undergoing single-level MIS-TLIF operations consisting of 32 NE-IFCs and 30 EXP-IFCs. There was no difference in latest follow-up time between EXP-IFC and NE-IFC group, respectively  followed by cages implanted at L5/S1  and those at L3/L4 .There were no significant differences in demographics between the two groups, as shown in p = 0.448) and EBL . No significant difference in operative time between the two groups was also observed . In all patients, the post-operative clinical function scores improved compared with the pre-operative scores, and these improvements lasted until the last follow-up. During the follow-up period, none of the patients required reoperation. No other differences in clinical function scores were observed between the EXP-IFC and NE-IFC groups  and at 1 year follow-up . In a typical case, x-rays reviewed at 1 week and 1 month postoperatively shown in figure 2 demonstrated effective improvement of the DH and FH compared to the preoperative period (p = 0.022) and at 1 year follow-up . Interestingly, for FH on the contralateral side, the patients who used the EXP-IFC had a significantly higher mean FH at post-operative  and at 1 year follow-up . No significant differences in LL were observed between patients with EXP-IFC and NE-IFC at 1 year follow-up . Similarly, different type of the implants did not contribute to changes in SL (Radiographic analysis showed that patients who use EXP-IFC in MIS-TILF had a significantly higher mean DH at post-operation (9.02 ± 0.5 mm EXP-IFC vs. 8.08 ± 1.08 mm NE-IFC, e period . After 1es in SL .For EXP-IFC used in MIS-TLIF, postoperative complications occurred in 3 cases, of which 1 case was cerebrospinal fluid leakage, which was cured by head and foot high position and dressing pressure bandaging. The other 2 cases had transient lower limb weakness after operation and were discharged from hospital after 1 month of nerve nutrition and enhanced functional exercise. For NE-IFC used in MIS-TLIF, Postoperative complications occurred in 3 cases: temporary aggravation of nerve root symptoms, which was related to intraoperative nerve root traction, improved after symptomatic treatment such as dehydration and detumescence, and mild low back pain remained in some cases after follow-up.Previous studies showed that restoration of sagittal alignment improve clinical results , 20. ThiInterbody fusion can rebuild the spinal stability and make the abnormal load correction, so the selection of intervertebral support materials was important in ensuring and improving the operation curative effect . MoreoveWhen DH is reduced to about 4 mm, especially with the loss of height at the posterior edge of the intervertebral space, the foraminal area volume decreases accordingly, which may cause nerve roots compression in the foraminal area . The useThe main complications of vertebral body fusion include nerve root or cauda equina injury, dural tear, intervertebral space infection, fusion cage displacement or prolapse, intervertebral bone graft non-fusion, loss of intervertebral space height and adjacent vertebral body degeneration instability and so on . In thisThere are several limitations in our study. First, election bias may exist, as the choice of EXP-IFC or NE-IFC wax`s largely determined by surgeon preference and patient pathology examination. Second, postoperative complications were evaluated by possible incomplete clinical records and possible selection or information biases were introduced. Third, the analysis of subsidence is performed by lateral x-ray measurements and will be strengthened by further radiological studies. Future studies will benefit from increased CT analysis of subsidence during and after operation. Fourth, the small sample size and limited incidence of events in our study limit the correlation between the results of the study and the entire population, and increase concerns about over-fitting of regression analysis. Finally, the mean follow-up time of 1 year is relatively short and long-term follow-up is needed to further explore the effects of EXP-IFC.In conclusion, EXP-IFC that support interbody fusion is less invasive than NE-IFC in MIS-TLIF and the use of EXP-IFC is better for the restoration of disc height and intervertebral foramen height, which caters to the current minimally invasive concept of spinal surgery. Thus, the increased cost of EXP-IFC maybe reasonable due to the pathology requirement of greater foraminal decompression. EXP-IFC has a good clinical application prospect, and is worthy of further research and application.


The PAS remained stable at a follow-up period of 1 year. In conclusion, BSSO advancement surgery could be regarded as a stable procedure for widening of the PAS with maintenance of positive space at follow-up.Lack of evidence exists related to the three-dimensional (3D) pharyngeal airway space (PAS) changes at follow-up after isolated bilateral sagittal split osteotomy (BSSO) advancement surgery. The present study assessed the 3D PAS changes following isolated mandibular advancement at a follow-up period of 1 year. A total of 120 patients  who underwent BSSO advancement surgery were recruited. Cone-beam computed tomography (CBCT) scans were acquired preoperatively (T0), immediately following surgery (T1), and at 1 year of follow-up (T2). The volume, surface area, and minimal cross-sectional area (mCSA) of the airway were assessed. The total airway showed a 38% increase in volume and 13% increase in surface area from T0 to T1, where the oropharyngeal region showed the maximum immediate change. At T1–T2 follow-up, both volumetric and surface area showed a relapse of less than 7% for all sub-regions. The mCSA showed a significant increase of 71% from T0 to T1 ( Dentofacial deformity in skeletal class II patients is most commonly characterized by mandibular retrognathism . One of Since the mid-1980s, numerous studies have been carried out assessing airway changes in patients undergoing mandibular advancement surgery ,7. With The main limitation of the aforementioned studies has either been related to their small sample size ,16 or shA total of 120 patients were recruited, consisting of 40 male and 80 female patients having a mean age of 26.0 ± 12.2 and a follow-up period of 12.0 ± 2.6 months. Data were collected from a period of August 2014 through March 2020. Patients who underwent isolated mandibular advancement surgery without genioplasty and had a craniocervical angle (N-S-Ba) of less than 5° for overcoming the variation in head position  were inc2 field of view, 96–110 kV, and a slice thickness of 0.3–0.6 mm. Following CBCT acquisition, scans were exported in DICOM  format. The T0 images were re-oriented and adjusted to the FH plane where required.Cone-beam computed tomography (CBCT) scans of the patients were acquired preoperatively (T0), immediately following surgery at an interval of 1–6 weeks (T1), and at 1 year of follow-up (T2). All scans were acquired using a standardized scanning protocol suggested by Stratis et al. , and patVoxel-based registration was applied utilizing the anterior cranial base for superimposition of T1 and T2 scans onto the T0 scan using Amira software . Following image registration, all data were imported to ProPlan CMF 3.0 , where segmentation of airway was performed with the initial threshold setting between −1024 to −500 Hounsfield units (HU). Additionally, manual adjustment was performed in cases where a proper depiction of the airway was not observed. Following segmentation, planes were reconstructed for dividing the PAS into the following anatomical regions: nasopharynx, oropharynx, hypopharynx, and complete PAS. The division of sub-regions was performed based on previously validated anatomical and technical limits proposed by Guijarro-Martínez and Swennen  22]. Th. Th22]. Thereafter, the DICOM data set was imported to InVivo Anatomage software  for determining the mCSA of the complete airway. It was defined as the minimal cross-sectional area along the airway axially extending from the posterior nasal spine superiorly until the anterior–inferior point of the body of the fourth cervical vertebrae inferiorly. The software automatically detected and calculated the mCSA . ValiditData were analyzed with MedCalc Statistical Software version 19.2 . Descriptive statistics including percentage, mean, and standard deviation were calculated for all the data. Normality of data was assessed with the Shapiro–Wilk test. For normally distributed data, a t-test was utilized to determine the change in PAS parameters from T0 to T1 and T1 to T2. A Wilcoxon signed-rank test was applied for data with non-parametric distribution. A Spearman correlation coefficient was calculated for assessing the relationship between the amount of advancement and mCSA change immediately after surgery and at follow-up. Statistical significance was set at 0.05 for all parameters.p > 0.062), thereby confirming the stability of head position. Patients underwent a mean mandibular body advancement of 5.7 ± 2.3 mm anteriorly and a relapse of −1.2 ± 1.2 mm posteriorly. No significant difference was observed in relation to the cranio-cervical angle (p < 0.0001) from T0 to T1. When divided into sub-regions, most of the significant volumetric changes occurred in the oropharyngeal region followed by the nasopharynx (p < 0.0001). No significant volumetric changes were seen in the hypopharyngeal region immediately following surgery (p = 0.948).p = 0.004) and oropharyngeal sub-region (p = 0.004) showed a significant decrease. No significant changes in surface area were observed at follow-up, with both total airway and all sub-regions showing an increase in surface area. According to p < 0.0001), whereas a non-significant relapse in the opposite direction was observed at T1–T2 .At T1–T2 follow-up, both volumetric and surface area showed a relapse of less than 7% for all sub-regions, where the total airway volume decreased by 5% and the surface area increased by 3%. Among the volumetric measurements, only the total airway volume (p < 0.0049)  . Both th 0.0049) . FurtherThe present study was conducted to address volumetric, surface area, and mCSA changes immediately after surgery and at a 1-year follow-up in a large group of patients. Our findings suggested a significant increase in the total PAS and mCSA immediately following surgery. The most likely explanation for this change could be related to the anatomical changes achieved by the BSSO advancement, where the most prominent change includes the elevation of the hyoid bone during surgery with supero-anterior movement due to the conjoint response of supra- and infra-hyoid muscles and change in tongue position . The oro- and hypopharyngeal region showed the highest change in volume immediately after surgery. This could be attributed to the stretching of the genioglossus and geniohyoid muscles, which originate from the mental spine and are responsible for protruding the tongue and hyoid bone anteriorly . These mAdditionally, a negligible to weak correlation existed between the PAS changes and skeletal changes at both immediate and follow-up time points, thereby also confirming that the skeletal relapse had a minimal influence on the PAS volume and dimensions. As no studies were found correlating skeletal movement and relapse with PAS changes in isolated mandibular advancement cases at follow-up, a comparison with similar literature was not possible. Nevertheless, AlSaty et al. also did not find any correlation between skeletal relapse and change in airway space following maxillomandibular advancement with and without genial tubercle advancement .The significant immediate increase in the airway dimensions and mCSA immediately after surgery was consistent with other studies ,12,15,29At follow-up, a significant decrease in the total airway volume was observed, where the oropharyngeal region showed maximal change. Even though mandibular advancement has been known to be a stable procedure, skeletal relapse of the distal segment is still observed in a posterior direction at follow-up. This posterior relapse is associated with recoiling of the hyoid musculature, which exerts force in a posterior direction, thereby acting as a precipitating factor for causing the oropharyngeal airway to relapse . Our finInstead of assessing the constriction in each segment separately, mCSA was assessed for the complete oro/hypopharyngeal airway. We believe that by doing so it provides more clinically relevant information about how the constriction changes for the complete airway instead of focusing on the segments separately for mandibular advancement surgery . A 71% iThe study had certain limitations. Firstly, the breathing movements during the CBCT scan acquisition were not controlled, which could have led to bias within our findings. However, the large sample size could have overcome these small inconsistencies. Secondly, the tongue movement was also not controlled at all the time points. In the midst of these limitations, we believe that our study provides a clinically relevant update on airway changes in patients undergoing isolated mandibular advancement. Further comparative studies should be conducted to assess PAS follow-up changes following single- and double-jaw surgeries and to assess whether airway changes differ between OSA and non-OSA patients.Isolated BSSO advancement surgery led to a significant, immediate increase in the total airway volume, surface area, and minimum constriction area. All these changes remained stable at a 1-year follow-up. Based on our findings, BSSO advancement surgery could be regarded as a stable procedure for widening of the PAS with maintenance of positive space at follow-up.


Legionella pneumophila outbreaks require organism culture. The ability to sequence L. pneumophila directly from clinical samples would significantly reduce delays. Here, we develop a method for targeted next-generation sequencing (NGS) of selected L. pneumophila genes utilizing a CRISPR/Cas9-based target enrichment system. We determine the method’s utility by typing cultured L. pneumophila isolates and subsequently apply the method directly to patient samples. We sequenced 10 L. pneumophila isolates by 2 methods, (i) whole-genome sequencing (WGS) and (ii) targeted (CRISPR/Cas9-based) finding low-abundance sequences by hybridization (FLASH)-NGS, sequencing 57 selected genes. The targeted NGS of 57 genes was more efficient than WGS, and phylogenetic analysis of the 57 genes yielded the same classification of the L. pneumophila isolates as that based on analysis of whole-genome data. Furthermore, targeted NGS of L. pneumophila performed directly on patient respiratory samples correctly classified the patients according to their corresponding cultured isolates. This provides proof of concept that targeted NGS can be used to sequence L. pneumophila directly from patient samples. Studies on a larger number of patient samples will further validate this method. Nonetheless, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms.Currently available methods for the laboratory investigation of IMPORTANCE The bacterium Legionella pneumophila is responsible for outbreaks of serious and life-threatening pneumonia called Legionnaires’ disease. There is a need for new molecular methods that allow investigation of Legionella outbreaks directly from patient samples, without the need for prior microbiological culture, which causes delays. Our study aims to address this problem. We have utilized a CRISPR/Cas9-based targeted next-generation sequencing (NGS) method that can be applied directly on human specimens. Furthermore, we show that analysis of the sequences of a small number of targeted genes offers the same classification of L. pneumophila as that based on data derived from the whole genome. Given the rising interest globally in sequencing pathogens directly from human samples, CRISPR/Cas9 targeted NGS methods have the potential to be widely applicable to microbial-outbreak investigations in the future, particularly in the context of difficult and slow-growing organisms. Legionella pneumophila is a cause of Legionnaires’ disease, a serious and life-threatening pneumonia. The inhalation of contaminated aerosols in the environment can lead to outbreaks of Legionnaires’ disease (LD) of public health significance. Timely typing of Legionella is important for outbreak investigation and management, but there is currently a lack of an accepted standard method for L. pneumophila typing. Over the past few years, with the wider availability and reduction in cost of next-generation sequencing (NGS), whole-genome sequencing (WGS) of Legionella isolates has become increasingly important in the investigation of point source outbreaks (–L. pneumophila genome (perhaps as few as 50 genes) could offer good discriminatory power and epidemiological concordance  directly from patient samples is difficult due to high levels of background human DNA and low target abundance. Methods like nested-PCR-based amplification of a smaller number of genes (7 genes) have been performed directly on patient samples -NGS   have beerculosis –17, and ngitidis . RecentlASH)-NGS , which uASH)-NGS . The metL. pneumophila. In this proof-of-principle study, we first tested the effectiveness of FLASH-NGS on bacterial isolates and then applied the method directly on human respiratory samples. The application of FLASH-NGS to Legionella typing will further demonstrate its versatility and pave the way for its wider use.Here, we have utilized FLASH-NGS for targeted sequencing of 57 genes and subsequent phylogenetic analysis of L. pneumophila isolates    to evaluAnalysis by both methods, core-genome SNP  and 57-gene SNP  phylogeny, based on 133,958 SNPs and 2,126 SNPs, respectively, produced results congruent with the published data  (The DNA extracted from 10 cultured isolates  was sequ samples . In cont samples . This re samples . For FLA t test) . A heatm t test) , except Of the 57 target genes, three genes  had consistently lower coverage in several isolates . Of inteL. pneumophila genomes were found to be highly related (isolates 3 and 4 and isolates 5 and 7) by both core-genome SNP and 57-gene SNP phylogeny (L. pneumophila reference strain Philadelphia (GenBank accession NC_002942.5). These relationships were evident in both WGS and FLASH-NGS data.Phylogenetic analysis of WGS data of the 10 isolates by core-SNP phylogeny and by 57-gene SNP phylogeny, based on 127,175 SNPs and 1,922 SNPs, respectively, yielded congruent results  and B. Fhylogeny , represeL. pneumophila pneumonia. These patients were designated 1P, 2P, 3P, 4P, 5P, and 7P and were the source of L. pneumophila isolates 1, 2, 3, 4, 5, and 7, respectively  values for these patients’ samples compared to the TC value for the sample from patient 1P (30 and 31 versus 20) , and hence, FLASH-NGS yielded only 197, 202, and 181 on-target reads, respectively, insufficient for further analysis.We attempted FLASH-NGS directly on respiratory tract samples from 6 patients who were hospitalized with culture-positive ectively . For patectively . For patectively . This corsus 20) . The samLegionella genes, 34 regions within 21 genes had the highest overlapping coverage in the samples from patients 1P, 3P, and 5P, with 50,079, 1,272, and 1,692 reads, respectively  and no reads were obtained for 2 genes (Ipg2494 and Ipg0689) . The latLegionella isolates. The 21-gene SNP phylogenetic analysis correctly identified the 3 patient samples as highly related to the corresponding cultured Legionella isolates 1, 3, and 5 .For the sample from patient 1P (which had adequate coverage of 53 genes), 53-gene SNP phylogenetic analysis correctly identified the sample as highly related to isolate 1, with 100% similarity. Isolate 1 had been cultured from the same clinical sample and was expected to be identical . Importantly, the removal of 4 genes from the analysis did not affect the overall phylogeny of the 10 isolates. We next compared phylogenetic analysis of sequence data covering the 34 regions within 21 genes that had the highest overlap for direct samples  and the 10 3, and 5 . The SNPL. pneumophila. We demonstrate that FLASH-NGS can effectively target and enrich genes of interest using DNA from L. pneumophila isolates. Furthermore, we show that these selected genes can be used to correctly classify  related and unrelated L. pneumophila isolates. Finally, and of significant clinical importance, we show that FLASH-NGS can be used to accurately facilitate phylogenetic analysis of L. pneumophila directly from human respiratory samples, without prior culture.This study assesses the role of targeted next-generation sequencing in phylogenetic analysis of L. pneumophila into genomically related and unrelated strains with good epidemiological concordance. This is demonstrated by the 57-gene SNP phylogenetic analysis of the public data set, which correctly identified L. pneumophila isolates involved in the main cluster of the outbreak . Similarly, in the 10 L. pneumophila isolates  contained very low bacterial loads and, not surprisingly, had insufficient coverage of targeted regions to allow phylogenetic analysis. This has been described elsewhere, such as in studies of direct whole-genome sequencing of Mycobacterium tuberculosis from respiratory specimens, which have shown that paucibacillary samples (as measured by smear or PCR) generated incomplete sequence coverage .L. pneumophila isolates from other parts of the world. Another limitation for targeted NGS methods includes the current lack of user-friendly bioinformatics tools for sequence data analysis, particularly for the purposes of bacterial typing. Many software packages are currently geared toward analysis of whole-genome data or are proprietary, with high licensing fees making access difficult. This limited our FLASH-NGS data analysis to SNP-based phylogeny. The development of easy-to-access software packages for bioinformatics analysis of targeted NGS data will be important for the development and utility of this method.There are some limitations of the FLASH-NGS system. We found that four genes had low coverage for the bacterial isolates and the patient samples. Although it is unclear why these genes had lower coverage, they were all small genes, with only 2 to 3 target guides per gene. An improvement in the initial design of the target guides by including additional targets per gene and thus building redundancy may be beneficial. In addition, mutations in critical sites may affect gene coverage. In isolates 6 and 9, we found a mutation in the PAM motif  , and this may be the cause of the low coverage in one gene. It would be of interest to assess how our method would perform on L. pneumophila directly from respiratory samples is possible using targeted FLASH-NGS. Future studies that incorporate a larger number of patient samples will help to optimize and validate this method further and determine its utility in diagnostic laboratory and public health investigation settings.This proof-of-concept study has demonstrated that phylogenetic analysis of L. pneumophila genome by a set of 20-bp CRISPR guide RNA molecules. At these target locations, Cas9 cleaves the DNA. The ends of the cleaved DNA molecules are not dephosphorylated and are therefore available for library preparation and attachment of adaptors. Amplification of the targeted regions and sequencing follows.An overview of the FLASH-NGS method used in this study is shown in the schematic in L. pneumophila isolates. This included 9 unique isolates from sporadic cases of legionellosis  identified at New South Wales (NSW) Health Pathology Microbiology Laboratory Liverpool between 2016 and 2019 and one well-defined control isolate (L. pneumophila Philadelphia strain ATCC 33152). FLASH-NGS was also applied directly to six patient samples. These samples comprised endotracheal tube (ETT) aspirates (2 patients) and expectorated sputum (4 patients) and were the sources for L. pneumophila isolates 1, 2, 3, 4, 5, and 7 included in the culture arm of the study. A summary of the samples used in this study is included in The FLASH-NGS method was applied to 10 Excess respiratory specimens were stored at 4°C for up to 1 week during initial diagnostic investigations and subsequently frozen at −80°C for 4 to 21 months. The sample from patient 2P was stored as a DNA extract from sputum at −80°C for 42 months.L. pneumophila that were part of several outbreak investigations in Sydney in 2016 (Table S1)   were useEthics approval by the SWSLHD Human Research Ethics Committee was obtained for accessing patient data and excess stored respiratory specimens.L. pneumophila culture isolates were grown on buffered charcoal-yeast extract (BCYE) medium  at 37°C for 5 to 7 days.The 10 Legionella PCR was performed using the 16-well respiratory pathogens B assay, version 8 . The assay is a two-step nested multiplex tandem PCR with an initial round of 15 cycles, followed by a second round of 30 cycles. Results are reported as TC take-off values comprised of second-round PCR TC values plus 15 cycles. The assay differentiates between L. pneumophila and Legionella longbeachae.L. pneumophila isolates using the QIAamp DNA blood mini kit (Qiagen) with a 3-h proteinase K digest at 56°C. The final DNA extract was eluted in DNase-/RNase-free water, and the quality was measured using the NanoDrop ND-1000 . DNA purity was deemed to be satisfactory at a 260-nm/280-nm ratio of 1.6 to 2.2 and a 260-nm/230-nm ratio of >1.8. DNA concentration was measured by using the Qubit double-stranded DNA (dsDNA) HS assay kit .Genomic DNA was extracted from 10 g for 10 min. Genomic DNA was extracted using the QIAamp DNA blood mini kit  with a 3-h proteinase K digest at 56°C. DNA was eluted in DNase-/RNase-free water, with quality and concentration measured as described above for L. pneumophila isolates.Endotracheal aspirate specimens from patients 1P and 7P and sputum specimens from patients 2P, 3P, 4P, and 5P were digested with Sputasol  at a 1:1 ratio and incubated for 10 min at 37°C, followed by centrifugation at 5,000 × L. pneumophila  were compiled from seven genes used for sequence-based typing (SBT) of umophila –23 and 5A were coL. pneumophila Philadelphia (GenBank accession no. NC_002942.5). The FLASHit software removes from consideration any guides that match human DNA, allows zero mismatches in the protospacer-adjacent motif (PAM) proximal bases, and allows 2 or fewer mismatches in the full 20-mer , from a pool of single-stranded DNA oligonucleotides  as previously described , according to the manufacturer’s instructions. DNA libraries were sequenced using the MiSeq system and the 2 × 100-bp kit, version 3 .L. pneumophila isolates. For the patient samples, the DNA inputs were 100 ng for the sample from patient P1 and 12.5 to 30 ng for the samples from patients P2, P3, P4, P5, and P7. The smaller DNA inputs for patients P2 to P7 were limited by the overall low DNA concentration in these samples. The FLASH-NGS protocol has been described previously  using the Burrows-Wheeler Aligner (BWA) (Reads were mapped to the reference genome of er (BWA)       of on 0.5.1 .L. pneumophila Philadelphia (GenBank accession no. NC_002942.5). Variant detection thresholds were set for a minimum coverage of 10, minimum mapping quality of 30, and minimum proportion of variant evidence of 0.75. Snippy outputs for the isolates were combined and analyzed by Snippy-core  using bcftools consensus  as described above. The SNPs within the 57 genes were extracted using SnpSift Intervals  Galaxy   and the general time-reversible (GTR) model with 500 bootstrap replicates.L. pneumophila isolates, reads were mapped to the reference genome of L. pneumophila Philadelphia (GenBank accession no. NC_002942.5) using BWA-MEM  (For the matches)  . Unmapped reads were discarded. Due to low coverage, the phylogenetic analysis was performed on fragments of 21 targeted genes. The coordinates for these gene fragments are included in Fig. S4. Variant calling was performed using Snippy  against the reference genome sequence. Variant detection thresholds were set for a minimum coverage of 4, minimum mapping quality of 20, and minimum proportion of variant evidence of 0.75. The rest of the workflow for SNP analysis of the 21 gene fragments in patient samples and 10 L. pneumophila isolates was as described for the 57-gene SNP analysis for WGS data. SNPs were also assessed by manual inspection of the read alignment (.bam) files against the reference sequence of L. pneumophila (NC_002942.5) in Integrative Genomics Viewer .For the analysis of patient samples, FASTQ files were imported into CLC Genomic Workbench version 7.5.5 and mapped to the reference L. pneumophila isolates, the workflow was as described for the 57-gene SNP analysis for WGS data.For the 53-gene analysis of patient sample 1P and the 10 PRJNA802331.Sequence data associated with this study have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA). The BioProject accession number is


PLGA-NPs were prepared by the single-emulsion solvent evaporation technique using dichloromethane (DCM)/chloroform as the organic phase with Polyvinyl-alcohol (PVA)/Polyvinylpyrrolidone (PVP) as stabilizers to encapsulate IND. The effects of different proportions of PVA/PVP with DCM/chloroform on the physiochemical properties  of the NPs were investigated. DSC was used to check the physical state, the possible complexation of PLGA with stabilizer(s) and the crystallinity of the encapsulated drug. Stabilizers at all concentrations produced spherical, regular-shaped, smooth-surfaced discrete NPs. Average size of 273.2–563.9 nm was obtained when PVA (stabilizer) with DCM, whereas it ranged from 317.6 to 588.1 nm with chloroform. The particle size was 273.2–563.9 nm when PVP was the stabilizer with DCM, while it was 381.4–466.6 nm with chloroform. The zeta potentials of PVA-stabilized NPs were low and negative (−0.62 mV) while they were comparatively higher and positive for PVP-stabilized NPs (+17.73 mV). Finally, drug-loaded optimal NPs were composed of PLGA (40 mg) and IND (4 mg) in 1 mL DCM/chloroform with PVA/PVP (1–3%), which resulted in sufficient encapsulation (54.94–74.86%) and drug loading (4.99–6.81%). No endothermic peak of PVA/PVP appeared in the optimized formulation, which indicated the amorphous state of IND in the core of the PLGA-NPs. The in vitro release study indicated a sustained release of IND (32.83–52.16%) from the PLGA-NPs till 72 h and primarily followed the Higuchi matrix release kinetics followed by Korsmeyer–Peppas models. The cell proliferation assay clearly established that the organic solvents used to prepare PLGA-NPs had evaporated. The PLGA-NPs did not show any particular toxicity in the HepG2 cells within the dose range of IND (250–500 µg/mL) and at an equivalent concentration of PLGA-NPs (3571.4–7142.7 µg/mL). The cytotoxicity of the hepatotoxic drug (IND) was reduced by its encapsulation into PLGA-NPs. The outcomes of this investigation could be implemented to prepare PLGA-NPs of acceptable properties for the encapsulation of low/high molecular weight drugs. It would be useful for further in vitro and in vivo applications to use this delivery system.A biocompatible, biodegradable and FDA-approved polymer [Poly lactic- A widoperties ,17,18,19operties . In addioperties ,22,23,24o/w) or double (w/o/w) emulsion solvent evaporation are the oldest and most extensively used techniques to encapsulate the hydrophilic and hydrophobic drugs into the PLGA-based micro- or nanoparticles [PLGA-NPs can be synthesized using different techniques, including emulsion solvent evaporation or diffusion, solvent displacement, salting-out, nanoprecipitation and many other techniques ,27,28,29articles ,9,22. Inarticles ,25. The articles ,31. The articles .Indomethacin (IND) is a nonsteroidal anti-inflammatory drug (NSAID) used to relieve fever, pain, swelling and stiffness of joints in moderate to severe arthritic conditions such as rheumatoid, osteo and gouty arthritis or spondylitis. It is also used for other purposes including in the shoulder pain affected by tendinitis or bursitis. It acts by the reversible inhibition of cyclooxygenase enzymes (COX-I and COX-II), also known as prostaglandin-endoperoxide synthase. The COX-1 catalyzes the synthesis of prostaglandins and thromboxane, while COX-2 is articulated in response to inflammation and injury. Thus, the antipyretic, analgesic and anti-inflammatory actions of IND take place as a result of decreased prostaglandin synthesis. Unlike other NSAIDs, IND also inhibits the enzyme phospholipase-A2 (causes the release of arachidonic acid from phospholipids). The antipyretic effect of IND might be due to its action on the hypothalamus causing vasodilation, increased peripheral blood flow and thus the dissipation of heat . The pooIn the present study, we investigated the effect of different formulation parameters on the physiochemical properties of the PLGA-NPs. We prepared four batches of PLGA-NPs using two different organic solvents, namely chloroform and dichloromethane (DCM), with two commonly used stabilizers, namely Polyvinyl alcohol (PVA) and Polyvinylpyrrolidone (PVP) and studied their influence on the particle size, its distribution and their morphological characteristics. We also investigated the effects of different concentrations and viscosities of the aqueous solution of the stabilizers on the PLGA-NPs. In addition, we utilized differential scanning calorimetry to check any polymer–stabilizer or drug interactions and the crystallinity of the encapsulated drug. To our knowledge, most of the literature has focused on the release behavior of the payload from the PLGA-NPs system. Thus, here we synthesized drug-loaded PLGA-NPs by the single emulsion solvent evaporation technique using IND as a model lipophilic drug. The physicochemical properties including the drug encapsulation, its loading and in vitro drug release and release kinetics were investigated in detail. Moreover, the toxicity of PLGA-NPs (with and without IND) was examined by performing the cytotoxicity using HepG2 cells.d,l-lactic-co-glycolic acid) (50:50) with molecular weight 40,000–75,000 and Polyvinylpyrrolidone  were purchased from Sigma Aldrich Co. . Indomethacin (IND) was procured from Winlab, UK. Polyvinyl alcohol (PVA) with 17,200 Mw was purchased from AVONCHEM Ltd. . Dichloromethane (DCM) and chloroform were purchased from PANREAC QUIMICA SA,  and MERK , respectively. Purified water was obtained through Milli-Q water purifier  system.Poly  equipped with UV-detector , binary pump , automated sampling system . The system was run by “Breeze software” . A C18 analytical column  was used at room temperature. The chromatography was performed by isocratic elution of the mobile phase composed of 75:25, v/v of Acetonitrile and Milli-Q® water (the pH of water was adjusted to 3.2 by Ortho-Phosphoric acid). The flow rate was 1 mL/min and the volume of injection was 30 μL. Total run time was 5 min and retention time (Rt) for IND was 3.5 min. The calibration curve was constructed in the concentration range 0.1, 0.2, 0.4, 0.8, 1.6, 2.5, 5.0 and 10 µgmL−1. The straight-line equation obtained was y = 150,383x − 6548.7 with a correlation coefficient (R2) of 0.9991.The reverse phase (RP) HPLC-UV method was used for the analysis of IND at 241 nm wavelength ,36,37. “o/w) single emulsion solvent evaporation technique [w/v) of polyvinyl alcohol (PVA) or polyvinyl pyrrolidone (PVP) by pulse sonication using a probe sonicator at 40% power for 40 s  using a probe sonicator  on an ice bath. The organic solvent was evaporated by magnetic stirring at 800 rpm at room temperature for overnight to obtain the suspension of NPs. The suspended PLGA-NPs were purified after washing with Milli-Q® water to remove the extra stabilizers and collected by ultracentrifugation at 15,000 rpm for 30 min at 4 °C . Finally, the drug-loaded PLGA-NPs were also prepared by the same procedure, where 4 mg of IND was dissolved into the organic phase containing PLGA. The washing process was performed three times for the purification and collection of PLGA-NPs. The obtained NPs were then dispersed in 10 mL of Milli-Q water, freeze-dried and were stored at −80 °C [The PLGA-NPs were prepared by the single emulsion solvent evaporation technique. Here, the PLGA-NPs were prepared using the oil-in-water . For zeta potential, by considering the dielectric constant of the dispersant , the electrophoretic mobility was calculated at 25 °C by the software. The electrophoretic values were utilized to estimate the zeta potential by the software DTS V-4.1  installed in the system. All the measurements were performed in triplicate.The hydrodynamic diameter (mean particle size), size distribution and zeta potential of the developed PLGA-NPs were checked by Dynamic Light Scattering (DLS) measurement by Zetasizer Nano-Series  as per the reported methods ,40. BefoThe shape and surface morphology of the PLGA-NPs was observed and determined by micrographs of the NPs obtained by Scanning Electron Microscopy (SEM)  using the gold sputter technique. The freeze-dried PLGA-NPs were coated with gold using an ion-sputter (Q150R Sputter unit) from Quorum Technologies Ltd.  in an argon atmosphere at 20 mA for 1 min. Observation and imaging was completed at 10 to 20 kV accelerating voltage and 8.5 mm of working distance. The magnification for the SEM images was kept at around 10 to 15 KX .−1 wavenumber range using OPUS software (V-7.8), Bruker Optics, Rosenheim, Germany.Attenuated total reflectance (ATR) FTIR spectrum of solid samples (of pure drug (IND), PLGA 50:50, Empty PLGA-NPs and IND-loaded PLGA-NPs-5) were recorded using Bruker ALPHA spectrometer . The spectrometer was equipped with a diamond prism with single bounce reflection ATR accessory. A thin layer of the solid sample was kept over the ATR accessory and the spectra were recorded from 4000–450 cm−1 for PVA and 360–460 for PVP s−1) at 25 ± 1 °C temperature.Viscosity study of the different concentrations of the stabilizers (PVA and PVP) was conducted to explore if there was a direct or indirect relationship between the viscosity and the characterization parameters . The visv/v) mixture of CHCl3 and DCM (to dissolve the PLGA and solubilize the IND) was added in the precipitant or pellet and vortexed. For complete extraction of the drug  from NPs, the mixture was subjected to ultrasonication  for 20 min. The resulting suspension was further centrifuged for 10 min at 13,500 rpm. The supernatant was collected and the concentration of the drug was analyzed by the reported HPLC-UV method at 241 nm wavelength [For the determination of the encapsulation efficiency (%EE) drug loading (%DL), 10 mg of drug-loaded PLGA-NPs were dispersed in 5 mL of Milli-Q water. The dispersion was centrifuged at 13,500 rpm at 10 °C for 15 min . The supernatant was removed and the sediments of PLGA-NPs (precipitant) were washed by Milli-Q water in triplicate with 2 min of vortexing. Thereafter, 10 mL 1:1  of sodium lauryl sulphate (SLS) was used as the release media. An in vitro release study was also performed using dilute HCl solution (pH 1.2) with 0.25% (w/v) of SLS as release medium. A total of 10 mg of freeze-dried drug-loaded NPs was put in 1 mL of PBS mixed by vortexing and transferred into dialysis tubing. Both the ends of the dialysis tubing were tied and put into 100 mL capacity beakers containing 50 mL of the release medium. Beakers were placed in a shaking water bath (100 opm) where the temperature of water bath was maintained at 37 ± 0.5 °C. At stipulated time points 1 mL of the sample was taken out from each beaker and centrifuged at 13,500 rpm for 10 min. The supernatants was collected and 30 µL of each was injected into the HPLC-UV system for the analysis of released drug [Based on the physicochemical features, two formulations from each batch were selected for the comparative in vitro release profile of IND from different PLGA-NPs. The in vitro release of IND from the selected and optimized PLGA-NPs was performed in in triplicate by the dialysis tubing method ,45,46. PR2) of kinetic plots obtained in different model equations, the release/diffusion-exponent  was calculated. The determination of n-values could provide an idea about the mechanism of drug release from the NPs [To check the kinetics and mechanism of IND release from the PLGA-NPs, the in vitro release data were fitted into different kinetic models ,48. The  the NPs ,49,50.. The n-value in the Korsmeyer–Peppas equation is known as the diffusion or release exponent.In the above equations, 2 gas at the rate of 20 mL per min. The results were analyzed by the software Pyris™  attached to the system. The thermal behavior of the materials used in the PLGA-NPs was examined at 10 °C per min of heating rate within 20 to 240 °C temperature range.The analysis of the physical state and possible complexation of the PLGA with the used stabilizers as well as the crystallinity of the encapsulated drug (IND) was checked through the differential scanning calorimetry technique through a DSC-8000  at the heating rate of 10 °C per min. Around 4–5 mg of PLGA-NPs was weighed and put in aluminum pans and the lids were crimped. The pans were hermetically sealed and placed in the sample cell of the calibrated DSC instrument to check the melting temperature of the materials ,51. BothA short-term stability on drug-loaded freeze-dried PLGA-NPs (IND-PLGA-NPs-5) was performed ,52,53. T2 (5%) at 37 °C using Dulbecco’s Modified Eagle’s Medium (DMEM), procured from UFC Biotech, Riyadh, Saudi Arabia). The media were accompanied with 1% mixture of “Penicillin-Streptomycin” , 10% Fetal Bovine Serum (FBS) and 1% L-Glutamine obtained from Alpha Chemika, Maharashtra, India and BioWest, Riverside, MO, USA, respectively, while the MTT was obtained from “Sigma Aldrich”, St. Louis, MO, USA.The human hepatic cancer cell (HepG2) lines were cultured under CO5 cells/mL. The suspension of cells were then transferred into flat-bottomed 96 well microplate in 100 μL medium and left overnight to attach. The medium was changed with 100 μL of fresh medium containing IND, IND-loaded NPs and empty PLGA-NPs-5 using the equivalent amount of NPs to obtain IND concentrations (5–1000 µg/mL) in the same media as the solvent. An equivalent amount of empty NPs was also used. The free IND was dissolved in DMSO and diluted with the media, keeping the concentration of DMSO not more than 1%, (v/v). Three wells were used as a negative control for cell viability, where only cells (without treatment) were suspended in the FBS free DMEM. Each product dilution was evaluated in triplicate. After 24 h, 48 h and 72 h of incubation, the cell suspensions were removed and the wells were washed with PBS. Then, 20 μL of MTT solution (at 5 mg/mL concentration in PBS) and 80 μL of the medium was added and incubated at 37 °C for 4 h. Subsequently, the culture fluid (containing media and MTT) was discarded leaving the precipitate of formazan crystals. The crystals were dissolved in 100 μL mixture (99.4 mL/0.6 mL/10 g) of DMSO, acetic acid and sodium lauryl sulfate at room temperature for 10–15 min. The microplates were observed and absorbance was measured at 570 nm using spectrophotometric microplate reader . The results were normalized as compared to the absorbance obtained for viable control cells (by considering as 100%) and expressed as plots of cells viability versus log concentration of IND. The IC50 values were determined for the employed cells against each product including the empty NPs using GraphPad Prism 5, San Diego, CA, USA.The in vitro cytotoxic activities of the free drug (IND solution) and the IND-loaded PLGA-NPs were investigated as compared to the empty PLGA-NPs-5 using HepG2 cells by assessing their viability through an MTT assay . The celn = 3). The data were expressed as mean with standard deviation (SD). Data were analyzed and compared by the Paired t-test using GraphPad Prism 5, USA. The p-value, less than (p < 0.05) was considered as statistically significant.All the experiments were performed in triplicate  making them better candidates for drug delivery applications. In this method, the emulsification of the two phases and the stabilization (droplet protection) of the NPs are prime factors, which are greatly influenced by the type of stabilizers used and their quantity. PVA and PVP were chosen as stabilizers because they facilitate the formulation of smaller PLGA-NPs with a spherical shape and a narrow granulometric distribution even at higher concentrations, in addition to their ability to prevent the coalescence of particles to aggregate ,56,57,58Using the proper preparation technique, such as single emulsion or double emulsion, is critical as the chosen method affects the physicochemical properties of NPs and the drug-loading capacity ,66. For The PLGA-NPs prepared by the single emulsion solvent evaporation method produced particles with an average size in the range of 273–563 nm when PVA was used as the stabilizer and DCM was used as the organic solvent, whereas it was in the range of 317–588 nm when chloroform was used as the solvent. As for PVP, the mean particle size was in the range of 287–684 nm when DCM was used as the solvent, while it was in the range of 381–466 nm when chloroform was the solvent .w/v) and using DCM as the organic solvent by keeping the other formulations factors constant , an increase in the size of the NPs was found from 273–563 nm (w/v) concentration of PVA when chloroform was used, which might be due to the strong anchoring of the hydrophobic segment of PVA with the matrix of PLGA which remained closely associated with polymer surface [Overall, the results of the particle size measurement indicate that PLGA-NPs prepared using PVA as stabilizer were smaller compared to PVP when DCM was used to solubilize the PLGA, with an exception at 1% PVP. This might be attributed to the good stabilizing property of PVP at this concentration (having optimum viscosity) with DCM. At the varying concentrations of PVA  which might be due to the comparatively low solubility/miscibility of DCM with water. On the other hand, when chloroform was used as an organic phase, it produced larger NPs. This contrasts with the previous work, where they reported that relatively smaller NPs were produced when they employed vitamin-E-TPGS as the stabilizer [As for the type of organic solvent, we observed that using DCM as the organic phase resulted in comparatively good-sized particles as compared to chloroform when NPs were prepared with the PVA at 1%, 3%, 6% and 9%, abilizer ,27,40,77abilizer ,57,58. Wabilizer ,78,79. Aabilizer  facilitaabilizer .Overall, PLGA-NPs prepared by the single emulsion solvent evaporation technique produced smaller NPs with a smooth surface and reliable properties when the most commonly used stabilizer (PVA) was employed, even at higher concentrations with DCM as the organic solvent, which is in agreement with previous reports ,82.w/v) of PVA, NPs obtained had a spherical and regular shape with a smooth surface (w/v) of PVA, however, NPs tended to aggregate and form irregular shapes  of PVP  of PVP . It is nThe FTIR spectra were recorded to investigate the possible interaction (if any) between PLGA and IND during the encapsulation of IND into the PLGA-NPs. The FTIR spectra of PLGA, IND, empty PLGA-NPs and IND-PLGA-NPs-5 are presented in −1 (aromatic C=C stretching and 1680 cm−1 (carboxyl stretching). The C–Cl stretching at 831 cm−1, C-O stretching ether at 1219 cm−1 and 1066 cm−1. Low intensity peaks at 2123 cm−1 and 2230 cm−1 wavenumbers were possibly indicate the O–H (carboxy) stretching [−1 was the characteristic peak of –C=O– stretching of aliphatic polyesters and also at 2030 cm−1 and 2124 cm−1 C=O bond stretching was observed. Moreover, the peaks at 727 cm−1 and 846 cm−1 were due to the C-H bending (vib). The IR scan of empty PLGA-NPs demonstrated characteristic bands at 1089 cm−1 and 1176 cm−1 which were due to the C-O stretching (ether) and peak at 1755 cm−1 was due to the aromatic C=C stretching. A less intense band appeared at 2023 cm−1 might be due to the –C=O– stretching of aliphatic polyesters (characteristic of PLGA) [−1 . The characteristic bands of IND did not appear in the drug-loaded NPs-5, which might suggest the proper encapsulation of IND into core of the NPs. Moreover, from this IR spectral analysis, it was concluded that IND was compatible with the PLGA (main excipient) used to prepare the NPs. However, the broadening and less intense bands appeared in the IR spectrum of IND-PLGA-NPs-5, also suggesting the absence of a chemical interaction between the drug (IND) and the polymer (PLGA).The sample of pure IND demonstrated the characteristic bands at 1585 cmof PLGA) ,87,88. Tw/v) of each formulation, was investigated using Milli-Q water as the solvent [In this study, the viscosity versus concentration relationship for PVA and PVP solutions at four different concentrations  and the viscosity of aqueous solutions of PVA and PVP increased with increasing their concentrations, exceptionally in case of 3% PVA as compared to 1% PVA. This might be due to the fact that viscosity is highly time and temperature-dependent. The decreased viscosity of 3% PVA solution might be attributed to the unwanted delayed processing in viscosity determination, also the temperature of the solution might be increased [The viscosity (Pa.s) of each solution was higher at the low shear rate , PLGA (40 mg) and IND (4 mg), DCM and CHCl3 were used as organic solvent(s) to dissolve the polymer and the drug. The developed formulations were further characterized.Considering the facts of the above findings about the empty PLGA-NPs, a few drug- loaded formulations were developed to encapsulate a model lipophilic drug . Where, PVA/PVP (1% and 3% The encapsulation efficiencies and drug loading capacities of the selected PLGA-NPs of the four batches (Batch-1 to Batch-4) are summarized in p < 0.05), a highest amount 52.16 ± 3.83% of drug was released from PLGA-NPs-5 (1% PVA), which was significantly (p < 0.05) higher from the rest of the three formulations of PVA group. The use of DCM as an organic solvent to prepare this formulation provided a good encapsulation of IND which might have resulted in its higher and sustained release in PBS with 0.25% SLS.A sustained release of IND from all the optimized PLGA-NPs was found at pH 7.4, as shown in 3 were almost same (15–16%) with no significant difference (p < 0.05), while a slightly improved release (19.4%) was found with the formulations prepared with DCM. However, the highest amount 51.6% of drug was released from PLGA-NPs-14 (3% PVP), which was slightly higher from the rest of the three formulations of PVP group. The higher concentration of PVP , provided better stabilization to this formulation, which might be the reason for improved and sustained release of IND in the selected release medium.At 12 h, the release of IND from PVP group formulations prepared with CHCl3), slightly less amount (40.06%) was released from PLGA-NPs-5 (1% PVA with DCM) and 39.2% was from PLGA-NPs-6 (3%PVA with DCM) which were significantly (p < 0.05) higher from the PLGA-NPs-2 (3%PVA with CHCl3) of PVA group formulations. Similarly, for PVP group formulations, the highest amount (43.16%) of drug was released at 72 h from PLGA-NPs-41 (3% PVP with DCM), slightly less (38.14%) was released from PLGA-NPs-9 (1% PVP with CHCl3) and an almost equivalent amount (32.5% and 35.81%) was released from PLGA-NPs-10 and PLGA-NPs-13, respectively.The in vitro release profiles of IND in acidic conditions (pH 1.2) from the selected PVA group and PVP group formulations of the four batches are illustrated in Overall, the cumulative amount of drug released from most of the PVA and PVP group PLGA-NPs in acidic release medium followed a biphasic release pattern as illustrated in The release of the drug at 12 h was very high at an acidic pH as compared to pH 7.4 as shown in v/v) SLS was used as release medium. Thus, only the release data obtained from this experiment was subjected to different kinetic models to check the mechanism of drug release from PLGA-NPs. The plots obtained in the applied models were represented in the k0 for zero-order, k1 for first order, HMk for Higuchi-Matrix, KPk for Korsmeyer–Peppas and HCK for Hixson-Crowell models) were summarized in R2) value, it was found that the selected NPs from the four batches primarily followed the Higuchi matrix release model, indicating the diffusion-controlled drug release from the PLGA-NPs, which was further substantiated by the mathematical calculation of the release-exponents  with 0.25% ; 0.994 and 0.2563 (Korsmeyer–Peppas model) as shown in n-values were lower than 0.5, yet, these values still indicated the diffusion-controlled release mechanism for IND from the PLGA-NPs [n-values were <0.45 in the present case of IND-loaded spherical PLGA-NPs, a Fickian-diffusion release mechanism was directed [k, indicating the rate of drug release) was directly proportional to the diffusion exponent. Therefore, the drug release was depended on the structural and physical properties of the IND and the PLGA-matrix. The highest values of “k”  were found with PLGA-NPs-5  to determine any possible complexation of the polymer (PLGA) with the stabilizers (PVA and PVP), and to check the crystallinity of the encapsulated model drug (IND). The overlay scans of DSC were represented in Although, no endothermic peak of stabilizer was noticed in the DSC scan of the lyophilized PLGA-NPs, indicating that the stabilizer was completely washed out during the purification steps by ultracentrifugation or the traces of the stabilizer (if remained), was not in the crystalline state. Furthermore, non-appearance of any new peaks throughout the scanning of the NPs, indicating the good compatibility among all the excipients used to develop the NPs.The results of the stability study  indicate50 were represented in Drug encapsulation into PLGA-NPs provides a good solution to minimize the potential toxicity while retaining the therapeutic efficacy of numerous loaded drugs including chemotherapeutics, analgesics and NSAIDs . Here, w50 were 242.4, 129.9 and 80.06 µg/mL at 24 h, 48 h and 72 h, respectively. A report of Xiong et al., 2013, indicated that the PLGA-NPs was not significantly toxic up to 300 μg/mL against RAW264.7 and BEAS-2B cells [50 were 430.5, 372.2 and 260.9 µg/mL at 24 h, 48 h and 72 h, respectively. Moreover, the cell proliferation assay conducted using empty-PLGA-NPs at equivalent concentration of IND  has shown IC50 values of 1096, 605.2 and 410.9 µg/mL at 24 h, 48 h and 72 h, respectively. Thus, even at these high concentrations of PLGA, the cells were viable at IC50 of 410.9 µg/mL when the duration of treatment was prolonged (72 h). This might be due to the time-dependent effect of PLGA-NPs on the cytotoxicity.The histograms of the percentage cell viability against varying concentrations of IND and the NPs were represented in 2B cells . Likewis50 of 7142.7 µg/mL. Thus, we can assume that even at this very high concentration of PLGA (empty PLGA-NPs/IND-PLGA-NPs-5), the product was non-toxic as even at these high concentrations of PLGA, the cells were viable and the IC50 was significantly reduced as the duration of treatment was prolonged , indicating the time-dependent effect of PLGA-NPs on the cytotoxicity.The MTT assay results clearly demonstrating the reduction in toxicity of IND by 66% in case of formulation (IND-PLGANPs) and empty PLGA-NPs with IC50 of cytotoxicity was also increased, which suggests that small sized NPs encouraged higher cytotoxicity at low IC50 values [At high concentrations the PLGA-NPs was toxic, this might be associated with the physicochemical properties including the particle size, zeta-potential and selective targeting of the PLGA-NPs. These play an important role in terms of its efficacy in biological systems. In previous reports, it was found that the increasing the particle sizes, the IC0 values ,105. The0 values ,104,105.5 cells/well, cell density of HepG2 cells. The highest live cells were observed at 72 h, and these results were similar to Miura et al., 1999, who seeded 4 × 105 cells/well in 6-well plates, and they believed that this was a more appropriate cell number for 6-well plates [The HepG2 are liver-derived human cell lines those display abundant parenchymal cell functions containing the metabolism of polycyclic aromatic hydrocarbons. The observed proliferation rate of HepG2  was measured, and it was found that these cells were doubled after 48.95 h at 5 × 10l plates . The doul plates ,108,109.Overall, the PLGA-NPs in the present investigation did not show any particular toxicity at most of the concentrations and could not influence the normal growth of HepG2 cells within the IND dose range (250–500 µg/mL) and at equivalent amount of PLGA-NPs (3571.4–7142.7 µg/mL). Conclusively, an ideal size, zeta-potential, dose, duration of treatment and targeting ability of PLGA-NPs to deliver the encapsulated drugs at desired site are essential to develop an efficient and safe drug carrier.p < 0.05) high IC50 value for the PLGA-NPs indicated that the FDA-approved polymer used to prepare the NPs was non-toxic. The above findings also indicated that the complete evaporation of the organic solvent (DCM) used to prepare the NPs. Moreoverthe cytotoxicity of hepatotoxic drug (IND) can be reduced by its encapsulation into PLGA-NPs, which was in agreement with previous reports [A significantly  high IC50 value for the NPs during the cytotoxicity study, indicated that the developed PLGA-NPs was non-toxic. Moreover, the adverse effect associated with the cytotoxic drugs such as IND (a hepatotoxic drug) could be overcome by their encapsulation into the PLGA-NPs. Nonetheless, further studies are needed to substantiate the current outcomes, which we are currently conducting in our lab that may further extend the utility of PLGA-NPs.We have acknowledged some process variables that are decisive to obtain proper sized, uniformly distributed PLGA-NPs with a smooth surface and a solid dense structure discrete. This study has shed light on the influence of preparation condition on physicochemical properties of PLGA-NPs, following the single-emulsion solvent-evaporation method to prepare the NPs. The PVA at lower concentrations was proved to be a good stabilizer to obtain an optimized PLGA-NPs with excellent size and surface properties, when DCM was used as organic solvent to dissolve the polymer. Controlling the particle size and morphology of the NPs may have a potential impact in tailoring the drug delivery applications of such nano-carriers. The developed and characterized PLGA-based NPs as controlled delivery system in the present investigation was stable and would have good potential applications for the loading of hydrophilic and poorly soluble lipophilic therapeutic agents. A short-term storage physical stability indicated that the optimized PLGA-NPs was stable at different temperatures for 30 days. A prolonged release of the IND from the optimized NPs suggested the sustained and controlled release property of PLGA. A significantly (